Cancer is the second leading cause of death in the U.S. Mutations in critical oncogenes, tumor suppressor genes and DNA repair genes contribute to tumorigenesis, prognosis and response to therapy. SpectraGenetics was founded to provide molecular diagnostics to impact cancer medicine. Peptide Mass Signature Genotyping (PMSG) will deliver sensitive, accurate, high throughput gene resequencing solutions to research and clinical markets that are demanding genetic biomarkers as integral parts of their early detection and drug development programs. This novel technology translates coding sequences into peptides for analysis allowing for high level multiplexing. Comprehensive analysis is achieved using a combination of translation in multiple reading frames and specific protease cleavages. The purpose of this SBIR project is to build a prototype automated RT-PMSG (Reverse Transcript-PMSG) process to provide analysis of target gene mRNA with a sensitivity that enables detection of variants that may be present as a minority component of the sample, as is often the case in a tumor nucleic acid sample. An added value of the multiplexing capability of RT-PMSG is that it allows for detection and analysis of splice variants present in an RNA sample and this capability will be exploited. Specifically, methods will be developed to produce peptide analytes in vitro using E. coli extracts for coupled transcription/translation reactions. A combination of proteases and multiple reading frames will be configured to analyze target mRNAs. The procedures for RT-PMSG will be developed using TP53 and FGFR3 mutant and alternatively spliced transcripts. Standard protocols will be automated, and validated, to achieve high- throughput, cost-effective genotyping. A high-throughput comprehensive resequencing platform that can sensitively analyze tumor-related genes in tumor nucleic acid samples will have wide application in research and clinical institutions. There is a great hope within the community that a greater understanding and characterization of tumor etiology, progression, and recurrence will enable prevention, early detection and improved treatments to reduce the pain and suffering caused by cancer. [unreadable] [unreadable]